P. Arcelli

GABAergic Neurons in Mammalian Thalamus: A Marker of Thalamic Complexity?

The present study evaluated the occurrence, distribution, and number of GABAergic neurons in the thalamus of different mammalian species (bat, mouse, rat, guinea pig, rabbit, cat, monkey, humans), by means of light microscopical immunoenzymatic localization of GABA or of its biosynthetic enzyme glutamic acid decarboxylase and by ultrastructural immunogold detection of GABA. Our data demonstrated that: 1) GABAergic local circuit neurons were detected in the thalamic visual domain in all the species analyzed, whereas in other thalamic nuclei their presence and number varied among species; 2) the number of GABAergic local circuit neurons progressively increased in the dorsal thalamus of species with more complex behavior; 3) the presence of local circuit neurons conferred a similar intrinsic organization to the dorsal thalamic nuclei, characterized by complex synaptic arrangements; 4) in the reticular thalamic nucleus, whose neurons were GABA-immunoreactive in all the examined species, the cellular density decreased from the bat to humans. These findings strongly suggest that thalamic GABAergic local circuit neurons are not directly related to the ability to perform specific sensorimotor tasks, but they are likely to reflect an increasing complexity of the local information processing that occurs at thalamic level.

Neuronal migration disorders and epilepsy: A morphological analysis of three surgically treated patients

Despite the increasing number of patients affected by neuronal migration disorders (NMDs) recently diagnosed in vivo by means of magnetic resonance imaging (MRI), few detailed data on the correlation between the neuroradiological and the anatomical features in the single NMD case are available. The present paper reports a combined cytoarchitectural and immunocytochemical analysis, by means of antisera recognizing specific neuronal and glial markers, of three MRI diagnosed NMD patients surgically treated for the relief of intractable seizures. The first case was a giant subcortical nodular heterotopia of morphologically normal neurons lacking any type of cortical lamination. The second case was a layered polymicrogyria with an abnormal amount of ectopic neurons in the underlying white matter. The third case was a focal cortical dysplasia characterized by a dramatic disruption of the normal cortical layering associated with marked cytological abnormalities. The present data demonstrate that the macroscopical and microscopical brain abnormalities can be markedly different in different NMD subtypes, and suggest that different anatomical substrates can underlie the intrinsic hyperexcitability of these brain malformations. The relevance of further prospective clinico-morphological studies for a better understanding of the mechanisms determining the development of these brain malformations is underlined.

Development of layer I of the human cerebral cortex after midgestation: Architectonic findings, immunocytochemical identification of neurons and glia, and in situ labeling of apoptotic cells

The development of layer I was studied in the human frontal cortex from 21 weeks of gestation (GW) to 2.5 postnatal months in series of adjacent sections processed for thionin staining, Bodian silver staining, and immunocytochemical labeling of neurons and glia. In addition, the terminal dUTP nick‐end labeling (TUNEL) method was used to label in situ DNA fragmentation. A progressive decrease of cell density and the disappearance of the subpial granular layer (SGL) appeared as distinctive developmental features of human layer I, consistently with previous investigations. The neuronal antigen microtubule‐associated protein2 was found to label preferentially Cajal‐Retzius cells and dendritic processes extending from the cortical plate. At midgestation, the calcium binding protein calretinin stained in the marginal zone numerous neurons, including the Cajal‐Retzius cells and their processes. Calretinin‐immunoreactive neurons decreased during the subsequent maturation: such decline was abrupt in the SGL, whereas bipolar calretinin‐immunopositive cells accumulated in the inner marginal zone to be presumably incorporated into the cortical plate. Cajal‐Retzius cells expressed calretinin throughout the examined developmental stages. The glial antigen vimentin was already expressed at midgestation, and vimentin immunopositivity decreased progressively in cell bodies and fibers of layer I during development. Glial fibrillary acidic protein‐positive elements gradually matured, and the positive cell bodies displayed the features of mature astrocytes at the end of gestation. Moreover, a decrease of free glial cells was observed in layer I, suggesting their progressive incorporation into the cortical plate. TUNEL‐positive cells were detected at midgestation in the marginal zone, and they were concentrated in the SGL until its disappearance; their number decreased dramatically throughout layer I after 30 gestational weeks. TUNEL‐positive nuclei or regressive changes were not detected in Cajal‐Retzius cells throughout the examined developmental stages. Thus, our data point out that naturally occurring cell death is an active mechanism contributing to the disappearance of the SGL but not to the subsequent developmental reshaping of human layer I, in which, instead, migratory phenomena should play a major role. In addition, our findings argue against a disappearance of Cajal‐Retzius cells due to regressive processes. J. Comp. Neurol. 410:126–142, 1999.

Calretinin immunoreactivity in the developing thalamus of the rat: a marker of early generated thalamic cells

The present work was aimed to study the immunocytochemical localization of the calcium-binding protein, calretinin, in the rat thalamus from embryonic day 14 to the third postnatal week. In the adult rat thalamus, calretinin immunoreactivity is intensely expressed in some intralaminar and midline nuclei, as well as in selected regions of the reticular nucleus. At embryonic day 14, calretinin was expressed by immature and migrating neurons and fibres laterally to the neuroepithelium of the diencephalic vesicle in the region identified as reticular neuroepithelium. At embryonic day 16, immunoreactive neurons were present in the primordium of the reticular nucleus and in the region of the reticular thalamic migration, where neurons showed the morphology of migratory cells. At the end of embryonic development and in the first postnatal week, calretinin-positive neurons were observed in selected region of the reticular nucleus and it was intensely expressed in some intralaminar and midline nuclei. Bands of immunopositive fibres were also observed crossing the thalamus. During the second postnatal week, the immunolabelling in the reuniens, rhomboid, paraventricular and central medial thalamic nuclei remains very intense while a decrease of immunoreactivity in mediodorsal, centrolateral and laterodorsal nuclei was observed. The immunostaining of fibres, particularly evident in the perinatal period, progressively decreased and it was no longer visible by the end of the second postnatal week when the distribution and intensity of calretinin immunostaining was similar to that observed in the adult rat thalamus. The present findings indicate that the immunolocalization of calretinin can be used to identify subsets of thalamic neuronal population during pre- and postnatal maturation allowing also the detection of the migratory pattern of early generated reticular thalamic neurons.

GABAergic interneurons in the somatosensory thalamus of the guinea-pig: A light and ultrastructural immunocytochemical investigation

This work was performed to confirm previous data reporting the presence of GABAergic interneurons in the ventrobasal complex of guinea-pig, and to investigate the intrinsic organization of this nucleus compared to that of thalamic nuclei lacking interneurons. Immunocytochemical experiments were performed on the thalamus of adult guinea-pigs perfused with mixed aldehydes using an anti-GABA serum. At light microscopy, the immunoreaction on floating Vibratome sections showed that GABAergic neurons are present only in the reticular and lateral geniculate nuclei and in the ventrobasal complex. Quantitative evaluation of their number indicated that they are 20 and 15% of the total neuronal population in lateral geniculate nucleus and ventrobasal complex, respectively, while they are less than 1% in ventrolateral nucleus. At the ultrastructural level, the postembedding immunogold procedure showed the presence, in the ventrobasal complex, of GABA-labeled profiles involved in complex synaptic arrangements similar to those found in carnivores and primates. Conversely, GABA-labeled terminals in thalamic nuclei devoid of interneurons formed exclusively axo-dendritic or axo-somatic contacts, like in rats and mice. The present data suggest that GABAergic neurons in the ventrobasal complex of guinea-pigs give rise to functionally important rearrangements of its intrinsic synaptic organization and that they represent the morphological basis for an intrinsic modulatory mechanism that is absent in other thalamic nuclei lacking inhibitory interneurons. The phylogenetic implications of these findings are also discussed in comparison to other animal species.

Postnatal development of gaba-immunoreactive terminals in the reticular and ventrobasal nuclei of the rat thalamus : A light and electron microscopic study

The postnatal development of inhibitory GABAergic circuits in the thalamic reticular and ventrobasal nuclei was studied in rats ranging from the day of birth to the end of the third postnatal week by means of a postembedding immunogold staining procedure to visualize GABA. In the reticular nucleus, GABA labeling was present from birth in cell bodies, dendrites, growth cones and a few synaptic terminals, whereas in the ventrobasal nucleus it was exclusively in axonal processes identifiable as growth cones, vesicle-rich profiles and synaptic terminals. In both nuclei, GABA-labeled synaptic terminals were, however, very scarce and immature in neonatal animals and they became numerous and morphologically mature only after the end of the second postnatal week. These findings suggest that inhibitory synaptic responses in the somatosensory thalamus are not yet fully mature throughout the first two postnatal weeks and support the hypothesis that GABA may initially play trophic roles. The relatively late maturation of the thalamic GABAergic system may have important functional consequences, as the reticulothalamic circuits are responsible for the generation of spindle wave oscillations whose cellular mechanisms are also involved in the generation of spike-and-wave (absence) seizures in humans and in animal models.

Ultrastructural features of the isolated guinea-pig brain maintained in vitro by arterial perfusion

The morphological features of cerebral tissue in the isolated guinea-pig brain maintained in vitro by arterial perfusion are described. Light and electron microscopic analysis of the thalamus, the somatosensory cortex and the limbic cortices (hippocampus, piriform and entorhinal cortices) was performed after different periods of incubation in vitro (1, 7 and 12 h), in parallel with an electrophysiological study. The morphological analysis showed that neuronal elements retained their normal appearance at both cellular and subcellular level in the examined brain regions up to an incubation period of 12 h. Immunoreactivity for GABA was also preserved for up to 12 h of in vitro perfusion. Vasogenic edema and perivascular extracellular swelling appeared after 7 h, together with signs of progressive astrocytic deterioration. These findings show that normal electrophysiological recordings correlate with good anatomical preservation of the isolated guinea-pig brain preparation after prolonged times of arterial in vitro perfusion.

Branching pattern of corticothalamic projections from the somatosensory cortex during postnatal development in the rat

In adult animals corticothalamic (CT) axons pass through the thalamic reticular nucleus (Rt) where they give off collateral branches innervating the Rt neurons. The postnatal development of CT projections from the somatosensory cortex, with particular reference to the branching pattern within Rt, ventrobasal (VB) and posterior (PO) nuclei, was investigated in the rat with anterograde tracing. Biotinylated dextran-amine (BDA) was iontophoretically injected into the somatosensory cortex of rats ranging from postnatal day (P) 0 to P30. At P1 most of the cortical axons traversed unbranched Rt and terminates in VB and PO, whereas at P3 they formed rudimentary branches in these nuclei. From P6 to P9 a progressive increase in the amount of dense clusters of terminal arborizations was evident in Rt, and by the second postnatal week more complex arborizations with a clear topographic arrangement were observed in Rt, VB and PO. Our findings indicate that CT fibers show a quantitative increase both in R1 and in somatosensory thalamic nuclei during the first postnatal week, although their terminal arborizations are however still incomplete. The pattern of collateralization of CT projections achieves an adult configuration at the end of the second postnatal week.

Ultrastructural immunolocalization of muscarinic acetylcholine receptor in the dorsal thalamus of rat

The ultrastructural distribution of muscarinic acetylcholine receptor (mAChR) in the dorsal thalamus of the adult rat was studied by means of pre-embedding immunocytochemistry using the monoclonal antibody M35. mAChR immunoreactivity (ir) was present with variable intensity in the different thalamic nuclei, but with a similar subcellular localization. Labeling was restricted to neuronal cell bodies and dendrites, where it was both in the cytoplasm and along the cytoplasmic side of the plasma membrane, in areas post-synaptic to small terminals with round clear vesicles but also in non-synaptic areas. Glial cells were unlabeled. By combining the pre-embedding immunostaining for mAChR with post-embedding immunogold labeling for GABA it was shown that GABAergic terminals made synaptic contacts with cholinoceptive structures, but no mAChR ir was present at their post-synaptic sites.

Ultrastructural characterization of the postnatal development of the thalamic ventrobasal and reticular nuclei in the rat

Electron microscopy has been employed to analyze the normal maturationai sequence that characterizes the postnatal development of synaptic circuits in the ventrobasal (VB) and reticular (Rt) thalamic nuclei of rats at different ages (from birth to the end of the third postnatal week). Throughout the first postnatal week, similar signs of immaturity are observed in both nuclei, mainly consisting in scarcity of cytoplasmic organelles, presence of wide extracellular spaces, and absence of myelinated fibers. Several synaptic terminals are however present from birth, thus indicating that some of the afferents have already reached and contacted their thalamic target during embryonic life. Most of the terminals are small and contain only a few round, clear vesicles, and therefore their cytological features do not allow the identification of their origin. In particular, in both nuclei, terminals with flat vesicles and symmetric specialization are only rarely observed, and in VB the ascending terminals are not distinguishable from terminals of other sources as they are in adults. During the second postnatal week, progressive maturationai changes in VB and Rt lead to neurons having well-developed cytoplasmic organelles and to an elaborate neuropil containing myelinated fibers and synaptic terminals that are morphologically heterogeneous and resemble the adult ones. The permanence of growth cone-like profiles and of numerous somatic and dendritic protrusions, often contacted by synaptic terminals, indicates that a certain degree of reorganization is still taking place in both nuclei. By the end of the third postnatal week the synaptic organization of VB and Rt is indistinguishable from that observed in adults. This ultrastructural study shows that the appearance of the neuropil of VB and Rt and the morphological complexity of the synaptic arrangements characteristic of the adult rat are not present in neonates, but are gradually acquired during the first three postnatal weeks, and that they result from progressive modifications in circuit organization involving both pre- and postsynaptic elements.